Cloning Protocol
Complete start-to-finish protocol for cloning shRNA constructs. This can also be used as a guide for any other kind of cloning. Please add troubleshooting questions/answers in the comments section! Annealing reaction make n +2 rxns *50uL total in the rows of 8 PCR tubes *1.5uL each primer (1.5 each of designed shRNA construct) = 3uL *5uL 10X NEBuffer 3 *42uL H2O *Add primer first, then 47uL of Buffer + H20 Master mix *Middle thermocycler: shRNA; or right most thermocycler: BZSH *90C for 4min *70C for 10min *0.1C/sec to 37C *4C hold Plasmid gel purification Cut your desired backbone vector w/ the appropriate restriction enzymes. (See RE digest). *Run gel, cut gel, put into eppendorf and weigh *Melt gel in (weight * 3) uL QG solubilizing buffer *Load into magenta column and spin 30sec @ max *Aspirate flow through *Wash column with 750uL PE (70% EtOH), spin 30sec @ max *Aspirate flow through—repeat if used a large volume of G buffer *Spin 30sec at max to dry column *Set column in a new tube, and elute with H2O *30uL H2O *Wait about 5 minutes *Spin 30 sec @ max *Nanodrop to quant; dilute in H2O to desired conc. Ligation This is the step where you glue your new gene (or shRNA construct) into the desired vector backbone and seal the new plasmid. Then it's ready to be amplified in bacteria! *Dilute annealed product: 2.5uL of product + 47u H2O *Always need vector only control (2uL H2O instead of dilute annealed product) *Dilute pSUPER plasmid: want a 10ng/uL solution *2uL of diluted annealed product *3uL plasmid mix (10-30ng total) *5uL 2X Ligase mix: *1uL 10X ligase buffer (must be warmed 20 mins before rxn)! → smell DTT to know buffer isn’t dead. *0.5uL T4 ligase (in cloning box) *3.5uL H2O *Add plasmid and annealed product first, then ligase mix *Mix! *Set at RT for 1-2hrs (60min minimum). Longer = more colonies *Large inserts: ligate O/N at 16C Transformation a. Thaw DH5a cells from -80 slowly on ice for ~10-20 mins b. Pre-chill ligation mixes on ice for ~10ish minutes c. Dry LB-Amp plates by propping open for 10 minutes d. Prewarm plates by placing in 37C incubator (by pcr room) i. Stacks of 4, staggered for even warming e. Flick to GENTLY re-suspend cells (DO NOT PIPETTE TO MIX) f. Add 45uL DH5a cells to each ligation reaction, flick to mix i. 45-50uL DH45a or stable2s + 5uL ligation mix g. Set on ice for 45 mins h. Turn on water bath to 42C i. Heat shock cells at 42C for 35 seconds Plate a. Return tubes to ice b. Pipette cell solution onto center of plate c. Add ~5 beads d. Rock DON’T SWIRL until cell solution is well distributed e. Set in 37C O/N (~14hrs) to grow i. Inverted! (agar side up). Don’t stack too high. Pick colonies grow up in 5mL in 1X LB-amp media O/N a. AMP is 1000X stock → 1uL amp per mL LB i. Use little tips p20 or p200 b. In shaking incubator c. Use pipette tip to select colony… d. Return plates to 4C, wrapped in parafilm Miniprep use 4/5mL of O/N liquid culture (save 1mL for glycerol stock) i. P1 buffer is at 4C and includes RNAse b. Spin down 2 tubes per sample of 2mL LB media in a 2mL centrifuge tube: 2 mins @ max, RT c. Aspirate super d. Re-suspend pellet in 250uL P1 buffer (pool the two tubes together, keep in one of the 2mL tubes) e. Add 250uL P2 (lysis) buffer, invert 4x to mix f. Add 350uL N3 neutralizing buffer and invert to mix (do in groups of 5) g. Centrifuge 10mins @ max h. Transfer super to blue tube from qiagen kit and centrifuge 30sec @ max i. Label with VWR pen (EtOH proof) i. Aspirate flow through j. Add 750uL PE buffer (70% EtOH) k. Spin 30 sec @ max l. Aspirate flow through m. Spin again to dry column: 30sec @ max n. Move column to a new prelabeled 1.5mL eppendorf o. Elute with 30uL water, sit 5minutes, spin 30sec @ max p. Nanodrop to quant, dilute to 250ng/uL i. 28uL @ Xng/uL → 28 * X = Y ng / (250ng/ul) = Z uL – 28uL = #uL to add Glycerol stock a. Mix 1mL of O/N liquid culture with 1mL of 60% glycerol in LB, freeze in dry ice and store in -80. Sequencing rxn 15uL total a. 500ng plasmid (2uL @ 250ng/uL) b. 8pmoles primer (2uL of 4uM stock) → pmol/uL = uM c. 11uL H2O d. Put in 8strip PCR tubes e. Elimbio.com—6pm pickup f. Ex: Seq Rxn example: i. Individual: 1. 2uL 250ng/ul plasmid (500ng) 2. 2uL 4uM primer (8pmol) 3. 11uL H2O 4. 15uL ii. 21 rxn master mix (for n=20) 1. 42uL 4uM primer 2. 231uL H2O a. 13uL /rxn b. Add the 2uL plasmid per rxn